More than 50% of patients with infective endocarditis (IE) develop an indication for surgery. Despite its benefit, surgery is associated with a high incidence of multiple organ dysfunction syndrome (MODS) and mortality, which may be linked to increased release of inflammatory mediators during cardiopulmonary bypass (CPB). We therefore assessed plasma cytokine profiles in patients undergoing valve surgery with or without IE.We performed a prospective case-control pilot study comparing patients undergoing cardiac valve surgery with or without IE. Plasma profiles of inflammatory mediators were measured at 7 defined time points and reported as median (interquartile). The degree of MODS was measured using sequential organ failure assessment (SOFA) score.p = 0.001); IL-18 (104.82 (105.99) vs. 57.30 (49.53) pg/ml, p<0.001); Mid-regional pro-adrenomedullin (MR-proADM) (2.06 (1.58) vs. 1.11 (0.53) nmol/L, p = 0.003); MR- pro-atrial natriuretic peptide (MR-proANP) (479.49 (224.74) vs. 266.55 (308.26) pmol/l, p = 0.028). IL-1β and TNF- α were only detectable in IE patients and first after starting CPB. Plasma levels of IL-6, IL-18, MRproADM, and MRproANP during CPB were significantly lower in survivors than in those who died.Between May and December 2016 we included 40 patients (20 in each group). Both groups showed similar distribution of age and gender. Patients with IE had higher preoperative SOFA  and operative risk scores . In-hospital mortality was higher in IE patients . Multiple organ failure was the cause of death in all non-survivors. At the end of CPB, median levels of following inflammatory mediators were higher in IE compared to control group: IL-6 (119.73 (226.49) vs. 24.48 (40.09) pg/ml, The presence of infective endocarditis during cardiac valve surgery is associated with increased inflammatory response as evident by higher plasma cytokine levels and other inflammatory mediators. Actively reducing inflammatory response appears to be a plausible therapeutic concept.NCT02727413.ClinicalTrials.gov, ID:  Infective endocarditis (IE) affects 1-10/100,000 persons per year and is associated with up to 40% in-hospital mortality . Surgica [Cardiopulmonary bypass (CPB) is an essential part of cardiac surgery for IE and has been shown to cause a systemic inflammatory response which may result in severe organ dysfunction and increased postoperative mortality  . In addi . Because , 11, andSuch data may provide valuable knowledge and may assist to develop measures aiming at perioperative reduction of cytokines and, thereby, may improve survival of endocarditis patients undergoing cardiac surgery.3, patients younger than 18 years-old, or pregnancy. We performed a case-control observational prospective pilot study on patients undergoing cardiac valvular surgery for either definite infective endocarditis according to the modified Duke criteria (20 patients), or VHD The study was approved by the ethics committee of the Jena University Hospital, Germany (reference number 4700-02/16).A written informed consent was obtained from each patient before inclusion in the study according to § 28 of the Declaration of Helsinki.Plasma profiles of inflammatory biomarkers: Procalcitonin, C-reactive protein, C-terminal proendothelin-1(CT-peoET-1), tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, IL-6, IL-10, IL-18.Plasma profiles of inflammation-related vasoactive mediators: midregional pro-Adrenomedullin (MR-proADM), copeptin pro-Arginine Vasopressin (CT-proAVP), midregional pro-Atrial natriuretic Peptide (MR-proANP).st and 2nd post-operative days, disclosed by Δ SOFA score as compared to pre-surgery status.Changes in organ dysfunction during the 1Use and duration of renal replacement therapy.Cumulative doses of concomitant medications  applied during the surgery and over 48 h thereafter.In-hospital mortality within 30 days post-surgery.Pre-operative data including assessment of surgical risk (EuroScore), pre-surgical co-morbidity (Charlson score) and acute organ dysfunction (SOFA score), operative data, and postoperative data including SOFA score, cumulative doses of concomitant medications, and use of renal replacement therapy were recorded in a computerized clinical research form (eCRF). Data acquisition was done via web application into the study management software OpenClinica®.Samples were collected at the following time points:12 to 24 hours before transfer to the operating theatreAt connection to the CPB60 minutes after connection to the CPB,disconnection of the CPB6, 24 and 48 hours after the end of the operation™ MR-proADM KRYPTOR™, B·R·A·H·M·S™ MR-proANP KRYPTOR™, B·R·A·H·M·S™, CT-proET-1 KRYPTOR™, B·R·A·H·M·S™ CRPus KRYPTOR™, B·R·A·H·M·S™ PCT sensitive KRYPTOR™ and B·R·A·H·M·S™ CT- proAVP KRYPTOR™ , respectively. The biomarkers IL-6, IL-1ß, IL-10, IL-18 and TNF-α (pg/mL) were measured using the IL-6 Human ProcartaPlex™ Simplex Kit, IL-1ß Human ProcartaPlex™ Simplex Kit, IL-10 Human ProcartaPlex™ Simplex Kit, IL-18 Human ProcartaPlex™ Simplex Kit, TNF-α Human ProcartaPlex™ Simplex Kit , in multiplexAll proteins were measured in EDTA plasma. The biomarkers MR-proADM (nmol/L), MR-proANP (pmol/L), CT-proET-1 (pmol/L), CRP (μg/mL), PCT (μg/L) and CT-proAVP (pmol/L) were measured using the B·R·A·H·M·SInformation on in-hospital mortality over 30 days was gathered from patient records or, in case of transfer to other facilities, through confidential inquiries conducted by the Principal Investigator.Data processing employed the software OpenClinica, which fulfils the regulatory requirements . Each subject was given an unambiguous patient identification number to ensure pseudonymized data analysisInformation on in-hospital mortality over 30 days was gathered from patient records or, in case of transfer to other facilities, through confidential inquiries conducted by the Principal Investigator.Data processing employed the software OpenClinica, which fulfils the regulatory requirements . Each subject was given an unambiguous patient identification number to ensure pseudonymized data analysis.0: p = 0.5 and the two-sided alternative is H1: p ≠ 0.5. For a sample size of 2 x 20 patients, our study has a power of ≥ 80% to detect p ≥ 0.75 (or p ≤ 0.25) at a two-sided significance level of 5% for the non-parametric test .Since there are no data from other trials for this patient population available a generic approach was used to estimate the required sample size. For the non-parametric sample size planning the effect size is given as relative treatment effect p . The nulth - 75th percentile) for continuous variables. For categorical variables, data were reported as frequencies and percentages. Mann-Whitney U tests were performed to compare continuous variables between two groups. Fisher’s exact test was used to compare in-hospital mortality rates of endocarditis and VHD patients. Spearman’s rank correlation was used to assess the association of cytokines and vasopressors. All analyses are exploratory and no adjustment for multiplicity was applied, the level of significance was set at 5% for each test. All statistical analyses were done using SAS 9.3 ).Baseline patient characteristics were reported as mean + standard deviation or median  or non-infectious valvular heart disease (n = 20).p <0.001), higher Charlson comorbidity index , and higher SOFA score (7 (IQR 5–7) vs. 4 (IQR 3–4.5), p< 0.001).p = 0.002). More than half of patients in VHD were operated using minimally invasive approach compared to only 15% in the IE group. The mean length of stay in the intensive care unit was longer in the IE group, however, the difference was not statistically significant . The in-hospital mortality was significantly higher in IE group .st post-operative day: 12 (9–13) vs. 6.5 (5.5–8.5), p<0.001; 2nd postoperative day: 10.5 (6–12) vs. 6 (6–7), p = 0.014). Changes between postoperative and pre-operative SOFA scores (ΔSOFA) within the same group were non-significantly different in both groups on the 1st post-operative day (5 (3–10) in the IE vs. 3 (3.5–10) in VHD, p = 0.173) as well as on the 2nd post-operative (3 (3–12) in IE vs. 3 (1.5–8) in VHD, p = 0.577).nd most common organ failure in patients with IE (40%), followed by cardiovascular failure (25%).st post-operative day in IE patients was substantially higher in IE patients compared to patients with VHD; however, the difference was not statistically significant. During the 2nd postoperative day, the cumulative dose of norepinephrine in IE patients was almost 5 times that in the patients with VHD (p = 0.009).There was no difference pre-operatively in norepinephrine doses between the two groups. The cumulative dose of norepinephrine during the 1p = 0.001)In In Table in Table in We found that MR-proANP correlated well with the post-operative cumulative doses of norepinephrine within 6h  and within 24 hours . MR-proADM correlated well with the post-operative cumulative doses of norepinephrine at 6h  and 24h . CT-proET1 correlated with the post-operative cumulative doses of epinephrine within 24h .Non-survivors had significantly stronger acute phase reaction to CPB  0.533, p = 0.019 and rs 0.574, p = 0.0127, respectively). The maximal level of PCT and MR-proADM correlated well with SOFA score on the 2nd postoperative day .We investigated the influence of surgical timing on mortality and on inflammatory markers. The mean time form diagnosis to operation was similar in survivors compared to non-survivors . Table in We also investigated the correlation between the duration of antibiotic therapy and the level of different cytokines and vasoactive peptides. There is no correlation between the duration of antibiotic therapy and inflammatory markers as shown in S4 TableOur results show that the presence of infective endocarditis during cardiac valve surgery is associated with increased inflammatory response as evident by higher plasma cytokine levels and other inflammatory mediators. Actively reducing inflammatory response appears to be a plausible therapeutic concept.In our study, IE patients undergoing valvular surgery had significantly higher mortality and higher multiple organ dysfunction (MOD) than patients with non-infectious valvular heart disease (VHD). IE patients required more post-operative norepinephrine therapy compared to the control group. In addition, the intensity of the acute phase response  and inflammmasome activation (IL-1β) and the release of vasoactive peptides (MR-proADM and CT-proET-1) after starting the CPB were stronger in IE patients compared to the VHD patients. Plasma levels of certain cytokines; CRP, PCT, IL-6, IL-10, or IL-18 or vasoactive peptides; MR-proADM or MR-proANP were significantly higher in non-survivors compared to survivors. Among IE patients, during CPB there was a correlation between maximal levels of IL-6, MR-proANP, or PCT and the post-operative SOFA score. Thus, we conclude that the stronger inflammatory reaction observed in IE patients during CPB may be responsible for the higher MOD and higher in-hospital mortality observed in this group of patients undergoing cardiac surgery.Surgical treatment is necessary in about 50% of IE patients and is associated with in-hospital mortality as high as 15–25% and 1-year mortality of 40% . The mecBustamante et al., measured pre-operative levels of IL-6, IL-8, and IFN-γ in patients with prosthetic vale endocarditis and found that they were linked to mortality . HoweverIn our study, we found that preoperative levels of IL-6, CRP, PCT, IL-18, IL10, MRproADM, and CTproET1 were higher in the IE patients compared to the control group.CPB initiates a systemic inflammatory response. Bernardi et al., reported that in their control group (patients undergoing elective cardiac surgery with CPB) the IL-6 was not detectable preoperatively and that median IL-6  increased to 63.6 pg/ml  at the end of CPB . In our Kellum et al., have shown that in patients with sepsis, mortality was higher in patients with high level IL-6 . In our In our study, IL-18 could be detected pre-operatively in both groups, however, its level was higher in IE patients compared to the control group. Venkatachalam et al., have shown that IL-18 is a key pro-inflammatory mediator in the pathogenesis and deterioration of patients with heart and vascular disease . This maIL-1β is a cytokine that plays critical roles in inflammation and cardiac dysfunction during severe sepsis . In our Elke et al., reported that high or increasing MR-proADM concentrations may help identify patients with a high risk of progression towards sepsis-related MOD. Our resIn both groups, plasma level of MR-proADM increased after starting CPB to reach its maximal level at 6 hours post-operative. Patients with IE had significantly higher levels of MR-proADM at each time point except at 24 and 48 h post-operative.Increased CT-proAVP concentration is described in several studies as a strong predictor of mortality in patients with chronic as well as acute heart failure , 24. CT-Natriuretic peptides (NPs) include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type NP. ANP andEndothelin-1 is a very potent vasoconstrictor which has inotropic and pro-inflammatory properties. Plasma ET-1 are increased in patients with CHF  and in pTNF-α is an essential component of the host immune response to infection and is responsible for the release of other pro- and anti-inflammatory mediators. TNF-α serum levels correlate with the severity of sepsis . TNF-α pIL-10 is one of the key anti-inflammatory cytokines as it decreases the production of inflammatory molecules, such as TNF-α and IL-6.In our study, IL-10 in both groups increased after starting CPB to reach its maximum at its end. Postoperatively, the decrease in the level of IL-10 was faster in the control group and its levels fell to the pre-operative level 24 hours post-operatively. While in the IE group, IL-10 did not fall to the preo-perative level even 48 h post-operatively. Currently, there is a consensus that imbalance between anti- and pro-inflammatory cytokines e.g. IL-6 and IL-10 is one of the cause of mortality in sepsis . Our resaureus as causative pathogen, and the severity of preoperative neurological complications are independent predictors of mortality[In our study, patients with IE had higher operative risk, measured by EuroSCORE II, compared to patients with VHD. The expected mortalityin IE patients, based on EuroSCORE II, was 19% and the observed mortality was 35%. EuroSCORE II does not include liver dysfunction or failure in its calculation. Howevermortality. All theCytokines secretion can be influenced by several factors  which may lead to bias in comparing different patients groups. Although operative procedures were similar in both IE and in VHD group and despite standardized operative and anesthetic techniques in our department, there might be some variations in techniques among surgeons and anesthetists. In addition, the limited number of patients is a partial limitation of this study.We could show in this pilot study that the inflammatory reaction to CPB, measured by different cytokines and vasoactive peptides, was stronger in patients with IE compared to patients with non-infectious VHD. We could also show that the magnitude of the inflammatory reaction correlated well with the degree of post-operative organ dysfunction. The results also showed that patients who died within 30 days of surgery had stronger inflammatory response than those who survived.S1 Checklist(PDF)Click here for additional data file.S1 Figp<0.05.This is the S1 Fig legend: Bright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; IL: interleukin; *: (TIF)Click here for additional data file.S2 Figp<0.05.Bright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; CRP: C—reactive protein; *: (TIF)Click here for additional data file.S3 Figp<0.05.Bright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; PCT: procalcitonin; *: (TIF)Click here for additional data file.S4 Figp<0.05.Bright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; MR-proADM: midregional pro adrenomedullin; *: (TIF)Click here for additional data file.S5 Figp<0.05.Bright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; proAVP: copeptin pro vasopressin; *: (TIF)Click here for additional data file.S6 FigBright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; CT-proET-1: C-terminal pro endothelin-1.(TIF)Click here for additional data file.S7 Figp<0.05.Bright line: non-survivors; dark line survivors;0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; MR-proANP: midregional pro atrial natriuretic peptide; *: (TIF)Click here for additional data file.S8 FigBright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; IL: interleukin.(TIF)Click here for additional data file.S9 Figp<0.05.Bright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; IL: interleukin;*: (TIF)Click here for additional data file.S10 Figp<0.05.Bright line: non-survivors; dark line survivors;0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; TNF: tumour necrosis factor;*: (TIF)Click here for additional data file.S11 Figp<0.05.Bright line: non-survivors; dark line survivors; 0 on the x-axis represents the beginning of cardiopulmonary bypass (CPB); the shaded area represents the CPB time; IL: interleukin;*: (TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(RTF)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file. 8 Oct 2019PONE-D-19-17603Changes in Inflammatory and Vasoactive Mediator Profiles During Valvular Surgery With or Without Infective Endocarditis: A Case Control Pilot StudyPLOS ONEDear Dr. Diab,Thank you for submitting your manuscript to PLOS ONE. 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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is a nice and interesting study about a tricky pathophysiological condition.Studying and profiling the "systemic scenario" of infective endocarditis and its potential operative and clinical consequenses is very challenging.In this specific investiagion, authors state that in presence of infective endocarditis during cardiac valve surgery, inflammatory response is much stronger than the one experienced by patients with non-infectious valvular heart disease.Even if cofounding factors can be numerous with such a background (some of them properly listed in limitations), this study could represent a meaningful trigger for further investigations, which are needed.Methodological process is well written and results are clearly presented.I would just suggest to provide some considerations about the potential role of surgical timing, combined with the use of antibiotics in limiting (or worsening) the inflammatory response.Secondly, I would better underline the impact of the use of vasopressors in perpetuating cytokines release involved in microcirculatory dysfunction.Finally, the limited amount of patients shoudl be mentioned as partial limitation as well.Reviewer #2: The article presents a prospective case-control analysis of 20 patients with endocarditis vs 20 pts with VHD without endocarditis having undergone CPB for cardiac valve surgery from May to December 2016 either isolated or combined.Endpoints:1. Plasma profiles of inflammatory biomarkers at pre-defined time points during the surgical intervention were measured: Procalcitonin, C-reactive protein, C-terminal proendothelin-1(CT-peoET-1), tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, IL-6, IL-10, IL-18.2. Plasma profiles of inflammation-related vasoactive mediators at pre- defined time points the course of the surgical intervention were: midregional pro-Adrenomedullin (MR6 proADM), copeptin pro-Arginine Vasopressin (CT-117 proAVP), midregional pro-Atrial natriuretic Peptide (MR-proANP).3. Changes in organ dysfunction during the 1st and 2nd post-operative days, disclosed by ΔSOFA score as compared to pre-surgery status.4. Use and duration of renal replacement therapy.5. Cumulative doses of concomitant medications  applied during the surgery and over 48 h thereafter.6. In-hospital mortality within 30 days post-surgery.For the study the authors collected preoperative, operative and postoperative data and blood samples for biomarkers at predefined times: - 12 to 24 hours before transfer to the operating theatre; - At connection to the CPB, - 60 minutes after connection to the CPB, - disconnection of the CPB - 6, 24 and 48 hours after the end of the operation.The two groups were significantly different for EuroSCORE II, SOFA score, COPD, PAVD, Admission type  or emergency (25%).The results showed a lower time of CPB in VHD group (no X clamp time is reported). Mortality was significantly higher in endocarditis group (35 vs 5%). Postoperative hemodialysis was similar.After that the authors reported all the figure legends and their results about the infiammatory results in a quite mechanical and confused way, please reorganize it and try to give us a more clear section.Also in the discussion section a lot of information about each cytokines and inflammatory markers in my opinion can be cut to make the speech lighter and more fluent.In the conclusions the authors state the inflammatory reaction to CPB was stronger in patients with IE group and that the magnitude of the inflammatory reaction correlated well with the degree of post-operative organ dysfunction.Some comments.Comment 1: the first two endpoints seem to be more a method more than an outcomes.Comment 2: is not very clear how the patients with endocarditis have a so high Euroscore. The authors has to better explain the patients preoperative status, surgical indication (emergency operation at least) and their preparation to operation in term of ABT weeks, blood cultures, time from diagnosis and embolic stroke (?) ). Moreover ther is a very high SD for the ES II of endocarditis group, in my opinion at least a comment of this findings is mandatory.Comment 3: In the operative data is not very clear the type of surgery for each patients. I suggest to divide the pts for the primary indication . No one patients needed a Bentall operation? Which prosthesis were used? All pts underwent full sternotomy? Please add Xclamp time.Comment 4: the mortality is quite high, probably a more specific explanation on the final cause of death can help us to understand the postoperative course; in my opinion is not very clear.Comment 5: In my opinion the conclusions are not very clear because is not well explained the correlation between clinical deterioration and inflammatory markers. I mean is not very clear which is the authors indication after their findings and what can we clinical improve in terms of indication and timing. Or at least just add a comment.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  9 Dec 2019Manuscript PONE-D-19-17603 Diab et al.Response to the Reviewers' CommentsWe would like to thank all reviewers for the constructive and helpful comments. We have taken care to revise the manuscript according to the critiques. We addressed below each comment point by point:We deposited our protocol in protocols.io with the following DOI: doi.org/10.17504/protocols.io.9esh3eedx.Journal Requirements:When submitting your revision, we need you to address these additional requirementsResponse: we checked that our manuscript meets PLOS ONE´s style requirementPlease provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained .Response:We clarified that in page 5 in the manuscript with track changesObtaining the informed consent:A written informed consent was obtained from each patient before inclusion in the study according to § 28 of the Declaration of Helsinki.Reviewer #1We wish to thank the reviewer for the kind and favorable commentsThis is a nice and interesting study about a tricky pathophysiological condition.Studying and profiling the "systemic scenario" of infective endocarditis and its potential operative and clinical consequences is very challenging.In this specific investigation, authors state that in presence of infective endocarditis during cardiac valve surgery, inflammatory response is much stronger than the one experienced by patients with non-infectious valvular heart disease.Even if cofounding factors can be numerous with such a background (some of them properly listed in limitations), this study could represent a meaningful trigger for further investigations, which are needed.Methodological process is well written and results are clearly presented.Comment 1: I would just suggest to provide some considerations about the potential role of surgical timing, combined with the use of antibiotics in limiting (or worsening) the inflammatory response.Response: We investigated the influence of surgical timing on survival and on inflammatory response. The mean time form diagnosis to operation was similar in survivors compared to non-survivors . There is a negative correlation between the time from diagnosis to surgery and IL-1 at 60 minutes CPB (Spearman´s Rank Correlation Coefficient (rs) -0.536, p=0324), TNF-α at 24h post-operatively , and IL-18 24h  and 48h postoperatively , which means the longer the time from the diagnosis to operation, the lower the level of aforementioned markers.We also investigated the correlation between the duration of antibiotic therapy and the level of different cytokines and vasoactive peptides. There is no correlation between the duration of antibiotic therapy and inflammatory markers. We added this information to the results section of the manuscript page 20 of the track changes manuscript and provided 2 tables S3 Table and S4 Table with the details of the Spearman correlation analyses for timing of surgery and for duration of antibiotic therapy as supporting information.Secondly, I would better underline the impact of the use of vasopressors in perpetuating cytokines release involved in microcirculatory dysfunction.Response: To clarify this point, we performed Spearman correlation analyses between the cumulative doses of vasopressor or catecholamine doses at 6 and 24h postoperatively. We found that MR-proANP correlated well with the post-operative cumulative doses of norepinephrine within 6h  and within 24 hours . MR-proADM correlated well with the postoperative cumulative doses of norepinephrine at 6h  and 24h . CT-proET1 correlated with the post-operative cumulative doses of epinephrine within 24h . We added this text to page 17 of the manuscript with track changes and submitted S2 Table with details of this analysis. Finally, the limited amount of patients should be mentioned as partial limitation as well.Response: We added the limited number of patients as partial limitation in page 26 of the manuscript with track changes.Reviewer #2: We wish to thank the reviewer for the constructive commentsThe article presents a prospective case-control analysis of 20 patients with endocarditis vs 20 pts with VHD without endocarditis having undergone CPB for cardiac valve surgery from May to December 2016 either isolated or combined.Endpoints:1. Plasma profiles of inflammatory biomarkers at pre-defined time points during the surgical intervention were measured: Procalcitonin, C-reactive protein, C-terminal proendothelin-1(CT-peoET-1), tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, IL-6, IL-10, IL-18.2. Plasma profiles of inflammation-related vasoactive mediators at pre- defined time points the course of the surgical intervention were: midregional pro-Adrenomedullin (MR6 proADM), copeptin pro-Arginine Vasopressin (CT-117 proAVP), midregional pro-Atrial natriuretic Peptide (MR-proANP).3. Changes in organ dysfunction during the 1st and 2nd post-operative days, disclosed by ΔSOFA score as compared to pre-surgery status.4. Use and duration of renal replacement therapy.5. Cumulative doses of concomitant medications  applied during the surgery and over 48 h thereafter.6. In-hospital mortality within 30 days post-surgery.For the study the authors collected preoperative, operative and postoperative data and blood samples for biomarkers at predefined times: - 12 to 24 hours before transfer to the operating theatre; - At connection to the CPB, - 60 minutes after connection to the CPB, - disconnection of the CPB - 6, 24 and 48 hours after the end of the operation.The two groups were significantly different for EuroSCORE II, SOFA score, COPD, PAVD, Admission type  or emergency (25%).The results showed a lower time of CPB in VHD group (no X clamp time is reported). Mortality was significantly higher in endocarditis group (35 vs 5%). Postoperative hemodialysis was similar.After that the authors reported all the figure legends and their results about the infiammatory results in a quite mechanical and confused way, please reorganize it and try to give us a more clear section.Response: We changed the figure titles and legends to meet the requirements of the PLOS ONE guidelines. Captions are not allowed to be included as part of the figure file or in a separate document.Also in the discussion section a lot of information about each cytokines and inflammatory markers in my opinion can be cut to make the speech lighter and more fluent.Response: We cut the unnecessary information in the discussion which does not serve our argument. In the conclusions the authors state the inflammatory reaction to CPB was stronger in patients with IE group and that the magnitude of the inflammatory reaction correlated well with the degree of post-operative organ dysfunction.Some comments.Comment 1: the first two endpoints seem to be more a method more than an outcomes.Response: We changed the text to:1. Plasma profiles of inflammatory biomarkers: Procalcitonin, C-reactive protein, C-terminal proendothelin-1(CT-peoET-1), tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, IL-6, IL-10, IL-18. 2. Plasma profiles of inflammation-related vasoactive mediators: midregional pro-Adrenomedullin (MR-proADM), copeptin pro-Arginine Vasopressin (CT-proAVP), midregional pro-Atrial natriuretic Peptide (MR-proANP)please see page 5 Endpoints in the main manuscript with track changes.Comment 2: is not very clear how the patients with endocarditis have a so high Euroscore. The authors has to better explain the patients preoperative status, surgical indication (emergency operation at least) and their preparation to operation in term of ABT weeks, blood cultures, time from diagnosis and embolic stroke (?) ). Moreover ther is a very high SD for the ES II of endocarditis group, in my opinion at least a comment of this findings is mandatory.Response: EuroSCORE II was high in the IE group because:• 30% of IE patients had previous cardiac surgery compared to 5% in VHD group• 75% of the IE patients underwent urgent operation and the rest underwent emergency operation, while all patients in VHD group were operated electively.• 50% of patients with IE were poorly mobile preoperatively while only 5% of patients in VHD group were poorly mobile.• 60% of IE patients had a critical pre-operative state compared to only one patient in the VHD group. • In addition, the presence of IE per se increases the EuroSCORE II. We added the factors we mentioned above to the pre-operative patient characteristics presented in Table 1 page 10 in the main manuscript with track changes.In addition we added a table with more pre-operative details of the endocarditis patients:S1 Table. We investigated the influence of surgical timing on survival and on inflammatory response. The mean time form diagnosis to operation was similar in survivors compared to non-survivors . There is a negative correlation between time from diagnosis to surgery and IL-1 at 60 minutes CPB (Spearman´s Rank Correlation Coefficient (rs) -0.536, p=0324), TNF-α at 24h post-operative , and IL-18 24h  and 48h postoperative , which means the longer the time from the diagnosis to operation, the lower the level of aforementioned markers.We also investigated the correlation between the duration of antibiotic therapy and the level of different cytokines and vasoactive peptides. There is no correlation between the duration of antibiotic therapy and inflammatory markers. We added this information to the results section of the manuscript and provided 2 tables S3 Table and S4 Table with the details of the Spearman correlation analyses for timing of surgery and for duration of antibiotic therapy as supporting information.We agree that the SD of the EuroSCORE II is much higher in the endocarditis group than in the VHD group. However, in order to compare the dispersion of the groups the coefficient of variance (CV) should be used, which is a standardized measure (SD divided by the mean). Since the mean is larger in the Endocarditis group than in the VHD group, the CVs are not much different . In addition, the greater standard deviation also reflects the expected greater heterogeneity of endocarditis patients compared to an elective patient population undergoing valve surgery. Comment 3: In the operative data is not very clear the type of surgery for each patient. I suggest to divide the pts for the primary indication . No one patient needed a Bentall operation? Which prosthesis were used? All pts underwent full sternotomy? Please add Xclamp time.Response: We added to Table 2 which shows Operative data and outcome in page 11 of the main manuscript with track changes information about the type of surgery in each group of patients, type of prostheses used, X-clamp time and the approach. We found that 55% of patients in the VHD group underwent minimally invasive surgery compared to only 15% in the endocarditis. This difference might cause bias in comparing the inflammatory reaction between both groups. In order to investigate whether the minimally invasive approach influences the inflammatory reaction or not, we compared the cytokine release in patients who underwent minimally invasive surgery compared to those who underwent full sternotomy within the VHD group. We found that for all markers to all time points, there was no significant difference between patient operated via minimally invasive approach and those operated via sternotomy, except for the median IL-10 at 24h postoperative which was higher in the sternotomy group (3.7 (IQR 6.32) vs. 2.20 (IQR 0.27). We added S5 Table to the supporting information showing the median and IQR of all markers to the defined time points in patients who underwent full sternotomy compared to patients operated via minimally invasive approach within the VHD group.In our study population no patient underwent a Bentall operation.Comment 4: the mortality is quite high, probably a more specific explanation on the final cause of death can help us to understand the postoperative course; in my opinion is not very clear.Response: In our study the cause of mortality in all non-survivors was post-operative multiple organ failure.As we have shown in Table 1, patients with IE had high EuroSCORE II 18.6�17.4 and high pre-operative SOFA score 6.895�2.58 which reached 11.263�2.579 on the 1st postoperative day. It has been previously shown that critically ill patients with SOFA score between 7-9 had a mortality of around 20%, and in patients who reached SOFA score 10-12 the mortality rateincreased to 40- 50%. Thus, in our high risk group of IE patients it was expected to have such a high mortality. Despite the expected high mortality, surgery is still recommended for such patients. We have previously shown that the in-hospital mortality in endocarditis patients who did not underwent cardiac surgery despite having an indication was 65%(3).. Comment 5: In my opinion the conclusions are not very clear because is not well explained the correlation between clinical deterioration and inflammatory markers. I mean is not very clear which is the authors indication after their findings and what can we clinical improve in terms of indication and timing. Or at least just add a comment.Response: We could show in this pilot study that the inflammatory reaction to CPB, measured by different cytokines and vasoactive peptides, was stronger in patients with IE compared to patients with non-infectious VHD. This conclusion was based on the following findings:• We have illustrated this in Fig 4A that IL-6 level at the end of CPB in the IE group was five-fold its level in the VHD group (119.73 pg/ml (226.49) vs. 24.48 pg/ml (40.09), p= 0.001). • I Fig 5A: Patients with IE had significantly higher levels of MR-proADM at each time point except at 24 and 48 h post-operative.• In Fig 5C Plasma levels of CT-proET-1 in IE patients were significantly higher p 24 h preoperative, at the beginning of CPB and 60 minutes thereafter• In Fig 5D: MR-proANP was significantly higher in the IE group during CPB and at 6h post-operatively• In Fig 6A In IE group after starting CPB median plasma level of IL-1β rapidly increased to reach a maximal level of 25.8 pg/ml and rapidly declined to approximately normal levels at the end of CPB. In the control group, IL-1β could not be detected at any time-point.• In Fig 7A: we showed that in both groups TNF-α was not detectable preoperatively. In the IE group its level increased immediately after starting CPB while in the control group it remained undetected at each time point.We could also demonstrate in Table 4 that the increase of levels of IL-6, PCT, and MR-proADM during CPB was associated with high SOFA score post-operatively. Thus, we can conclude that the magnitude of the inflammatory reaction correlated well with the degree of post-operative organ dysfunction. As SOFA score is the best score to report organ dysfunctions in sepsis patients as recommended by the Task Force for sepsis (4), we consider SOFA score an accurate tool to measure and report clinical deterioration. In addition, we also demonstrated in supporting information S1-11 Figs that Non-survivors had significantly stronger acute phase reaction to CPB  after starting CPB. Plasma levels of MR-proADM  were also higher in non-survivors to almost all time points compared to survivors. The difference was statistically significant from the beginning of CPB until 6 hours post-operative. MR-proANP plasma levels  were higher to all time points in non-survivors compared to survivors. The differences were statistically significant at 60 minutes after starting CPB until the 6 hours post-operative. IL-18 plasma levels  to all time points were substantially higher in non-survivors compared to survivors and differences became statistically significant 60 minutes after starting CPB until 6 hours post-operative. Plasm levels of IL-10 increased similarly in non-survivors and survivors after starting CPB, however, it remained significantly higher to all post-operative time points in non-survivors compared to survivors . Thus, we can conclude that patients who died within 30 days of surgery had stronger inflammatory response than those who survived.The aim of this study was to compare, for the first time, the peri-operative cytokines profiles of patients undergoing cardiac surgery for IE with those of patients with non-infectious valve disease. Although we gathered information about the pre-operative neurological disorders and the timing of surgery in the CRF of the study, these were not the main objectives of this pilot study. We hope that this revised version now meets your expectations and thank you and all the reviewers for your efforts that improved this manuscript. References 1. Vincent JL, de Mendonca A, Cantraine F, Moreno R, Takala J, Suter PM, et al. Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: Results of a multicenter, prospective study. Crit Care Med. 1998;26(11):1793-800.2. Ferreira FL, Bota DP, Bross A, Melot C, Vincent JL. Serial evaluation of the SOFA score to predict outcome in critically ill patients. Jama-J Am Med Assoc. 2001;286(14):1754-8.3. Diab M, Guenther A, Sponholz C, Lehmann T, Faerber G, Matz A, et al. Pre-operative stroke and neurological disability do not independently affect short- and long-term mortality in infective endocarditis patients. Clinical research in cardiology : official journal of the German Cardiac Society. 2016;105(10):847-57.4. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA : the journal of the American Medical Association. 2016;315(8):801-10.AttachmentResponse to Reviewers.docSubmitted filename: Click here for additional data file. 13 Jan 2020Changes in Inflammatory and Vasoactive Mediator Profiles During Valvular Surgery With or Without Infective Endocarditis: A Case Control Pilot StudyPONE-D-19-17603R1Dear Dr. Diab,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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In this study, resveratrol-loaded solid lipid nanoparticles (Res-SLNs) were successfully designed to treat MDA-MB-231 cells. The Res-SLNs were prepared using emulsification and low-temperature solidification method. The Res-SLNs were spherical, with small size, negative charge, and narrow size distribution. Compared with free resveratrol, the Res-SLNs displayed a superior ability in inhibiting the proliferation of MDA-MB-231 cells. In addition, Res-SLNs exhibited much stronger inhibitory effects on the invasion and migration of MDA-MB-231 cells. Western blot analysis revealed that Res-SLNs could promote the ratio of Bax/Bcl-2 but decreased the expression of cyclinD1 and c-Myc. These results indicate that the Res-SLN may have great potential for breast cancer treatment. Breast cancer is the most common malignant tumor among women worldwide. The main carcinogenic factors—including genetic mutations, endocrine disorders, and decline in immune function—may increase the risk of developing breast cancer ,2. AlthoResveratrol  is a natural substance which contains a stilbene class structure of flavonoid polyphenol compounds  [5]. In The development of drug delivery system in cancer treatment has attracted many researchers interest in recent years. Solid lipid nanoparticles (SLNs) have been under consideration for drug delivery system because they possess good physicochemical properties, have the possibility of modulating drug release, and can be metabolized by a variety of organisms. In addition, the SLNs can better protect the encapsulated drugs in the biological circumstances and offer better physiochemical features . In our Herein, the objective of this study was to design Res-loaded SLNs (Res-SLNs) for breast cancer treatment. Particle size, entrapment efficiency, zeta potential, UV–vis spectra and XRD pattern of the Res-SLNs were investigated. Furthermore, the anti-cancer effect of Res-SLNs was evaluated on MDA-MB-231 human breast cancer cells.Res-SLNs were prepared using emulsification and low-temperature solidification methods.The shape and morphology of resveratrol-loaded SLNs were observed by transmission electronic microscopy (TEM). As shown in UV–vis was taken to confirm the presence of resveratrol in Res-SLNs. As shown in The X-ray diffraction technique was employed to confirm the XRD pattern of Res, SLNs, and Res-SLNs. As shown in 50) was calculated. At various concentrations, as presented in 50 value was estimated at 40.82 ± 3.92 μg/mL for Res-SLNs-treated group and 72.06 ± 7.85 μg/mL for resveratrol-treated group, respectively. It is evident that Res-SLNs exhibited obviously more effective cell cytotoxicity in comparison with free resveratrol.The cytotoxicity of resveratrol and Res-SLNs against MDA-MB-231 breast cancer cell was investigated by SRB assay and the half inhibition concentration (ICHoechst staining was used to measure cell apoptosis. The presence of apoptotic nuclei was demonstrated by staining with Hoechst 33342 in MDA-MB-231 cells. As shown in The effects of resveratrol and res-SLNs on cell migration in MDA-MB-231 cells were determined by a scratch wound method. As shown in p < 0.05) and the Bcl-2/Bax ratio was decreased. Conclusively, the res-SLNs exhibited a more effective ability than free resveratrol in inducing cell apoptosis, probably because the water solubility property of resveratrol was increased when it incorporated into SLNs.Since Bcl-2 family members play a major role in apoptosis, the expression of Bax and Bcl-2 in MDA-MB-231 cells were analyzed by the treatment of SLNs, resveratrol and Res-SLNs. As presented in p < 0.05) and c-Myc (p < 0.01). These data suggested that res-SLNs could regulate the cell cycle by inhibiting the expression of cyclin D1 and c-Myc.Next, the effects of resveratrol and res-SLNs on the expression of c-Myc and cyclin D1 in MDA-MB-231 cell were examined by Western blotting. Recent studies have demonstrated that cyclin D1 and c-Myc were important target genes of the Wnt signaling pathway. c-Myc acts as an oncogenic transcription factor. The elevation of c-Myc level markedly increases both metastasis and invasion capabilities of tumors. Cyclin D1 regulates cell cycle by controlling G1/S transition, and the upregulation of cyclin D1 contributes to multiple cancers. As shown in Nanomedicine provides an excellent platform for drug delivery of anticancer agents in order to enhance their targeting ability and bioavailability. Some of the potential drug delivery systems are micelles, liposomes, polymeric nanoparticles, and solid lipid nanoparticles (SLNs) ,16,17,18In this study, resveratrol was successfully incorporated into SLNs. Res-SLNs was developed using an emulsification and low-temperature solidification method. Most of the materials for preparing SLNs are low cost. The lipids and surfactants are the two main materials used in SLNs. Stearic acid, a saturated monoacid triglyceride, was used as a negatively charged solid lipid, and Myrj52 as negatively charged surfactant to increase the chance of drug encapsulation. The heating of the system was found to be very important during the melting and mixing of the solid lipid. Upon cooling, pre-emulsion has smaller droplets, which may result in smaller nanoparticles. It was observed that the cellular uptake and distribution of SLNs were influenced by the particle size, surface charge, type of lipids, and the concentration of the emulsion ,20. TEM 50 of Res-SLNs was lower than that of free Res. Blank SLNs did not show any significant toxicity to the cancer cells. As we know, the bioavailability of resveratrol is typically very low. This is due to its poor solubility in water and fast pre-systemic metabolism. Formulating resveratrol in polymeric or lipid based delivery systems  is one possible strategy to overcome the drawbacks of the drug. The enhanced anti-cancer effect of Res-SLNs compared to free Res may contribute to the lipophilic nature of the carrier which facilitates the intracellular uptake. Apoptosis is one form of programmed cell death that plays a pivotal role in cancer therapy. Bcl-2, as the nodal point at the convergence of multiple pathways with broad relevance to oncology, is well known for its ability to suppress apoptosis  grade) were purchased from Sigma-Aldrich Corporation .Resveratrol  was purchased from Aladdin . Stearic acid, lecithin chloroform, and TweenResveratrol-SLNs were prepared using the solvent injection method. Briefly, Resveratrol (150 mg), stearic acid (200 mg), and lecithin (100 mg) were dissolved in 10 mL chloroform in a glass flask (as organic phase). Myrj 52 was dissolved in 30 mL distilled water and heated to 75 °C ± 2 °C in a water bath (as aqueous phase). The organic phase was injected into the hot aqueous phase under mechanical agitation at 1000 rpm, and the resulting solution was kept at 75 °C with the same agitation speed to remove the organic solvent. The condensed solvent (approximately 5 mL) was then transferred into an equivalent amount of cold water (0–2 °C) under continuous mechanical stirring (1000 rpm) for 2 h. The resultant suspension was centrifuged at 20,000 rpm  to remove the supernatant. The pellet was suspended in ultrapure water, refrigerated, and lyophilized. A drop of diluted solution of the Res-SLNs was placed in carbon-coated copper TEM grid , the samples were imaged using a JEM-1230 transmission electron microscope  and visualized at 120 kV under microscope. The particle size and the zeta potential of Res-SLNs are determined at 25 °C by photon correlation spectroscopy .The amount of entrapped Res was determined by UV–vis spectroscopy. A sample containing a determined weight of Res-SLNs was dissolved in the determined volume of ethanol. After the sample was completely dissolved, the concentration of Res was determined with a UV–vis spectrophotometer. The selected wavelength for Res measurement was 304 nm.The UV–vis spectra  were observed to investigate the possible chemical interactions. The absorbance spectra of free Res, Res-SLNs (dissolved in ethanol), and SLNs were measured using a Cary 50 UV–vis absorbance spectrophotometer. X-ray diffraction measurements were performed in order to characterize the crystallographic structure of the resveratrol-SLNs, resveratrol, and SLNs. The patterns were carried out with an X-ray diffractometer  in the range of 5°–50°. The measurements were performed at a voltage of 40 kV and 25 mA.2 incubator.The MDA-MB-231 (Human breast cancer) cells were obtained from ATTC. The cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum and 1% antibiotic solution and maintained at 37 °C in a 5% CO5 per well) were seeded in 96 well plates and incubated for 24 h in a 5% CO2 incubator. Afterwards, media was replaced with fresh media containing different equivalent concentrations of free Res and Res-SLNs and incubated for 24 h. After incubation, the cells were fixed in ice-cold trichloroacetic acid (10% w/v) for 1 h. The cells were washed and stained 0.4% SRB dye for 30 min at room temperature. The excess SRB dye was removed and 100 μL of 10 mM Tris buffer (pH 10.5) was added. The optical density of each well was measured at 515 nm on a Bio-Rad 550 ELISA microplate reader. All experiments were performed at least three times. The sulforhodamine-B (SRB) assay was performed to assess the potential antiproliferative effects of Res, Res-SLNs, and SLNs on MDA-MB-231 breast cancer cells. The cells  was added and incubated at 37 °C for 20 min in the dark. Thereafter, the dye liquor was removed, and the cells were washed three times with PBS. A fluorescence microscope  was used for the examination of each well at different fields of view.MDA-MB-231 cells were seeded into 6-well plates at a cell density of 1 × 10Apoptosis were assayed using the multifunctional Muse Annexin V and Dead Cell kit  according to the user’s guide and the manufacturer’s instructions. Briefly, the breast cancer cells were harvested and washed with PBS twice. Then, 5 μL of FITC-labeled enhanced annexin V and 5 μL (20 μg/mL) of propidium iodide were added to a 100 μL cell suspension. After incubation in the dark for 15 min at room temperature, the samples were immediately analyzed by Muse Cell Analyzer . All experiments were performed in triplicate. The cell cycle distribution analysis was measured using a Muse Cell Cycle Assay Kit (Merck-Millipore) according to the manufacturer’s instructions. Cells were trypsinized, washed with PBS and fixed in 70% ethanol overnight. Then, cells were centrifuged, washed with PBS, and analyzed by Muse Cell Analyzer .The breast cancer cells were seeded in 6-well plate until the cells reached to 90–95% confluency. The scratch wound was generated in the surface of the plates using a pipette tip in cells with Res, SLNs, and Res-SLNs treatment. Photographic images were taken at 0 and 24 h. The images were then analyzed using Image J software .The migration of cells was conducted using a 24-well transwell chamber  with gelatin-coated polycarbonate membrane filter. The invasive capacity of cells was performed using transwell precoated with matrigel . After incubation for 24 h, the upper surfaces of the transwell chambers were scraped with cotton swabs, and the migrated and invaded cells were fixed with 4% paraformaldehyde, and then stained with Giemsa solution. The stained cells were photographed.6 per well) and treated with Res and Res-SLNs (40 μM) for 48 h. After drug treatment, cells were harvested and resuspended in lysis buffer. All the samples were then centrifuged at 12,000 rpm at 4 °C for 10 min. The protein concentrations of cell lysates were determined using the bicinchoninic acid (BCA) protein assay kit . Total proteins (20 μg) were subjected to 10% acrylamide SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% nonfat milk in PBS with Tween 20 (PBST) at room temperature for 1 h. The membranes were probed with specific primary antibodies against Bcl-2, Bax, c-Myc, and CyclinD1  overnight at 4 °C. After incubation with the secondary antibody for 1 h at 20 °C, membranes were washed five times. The immunoreactivity bands were detected by ECL detection system. Quantification of protein bands was performed using the Image J sofeware .MDA-MB-231 cells were seeded in six-well plates . Statistical analysis was performed using the one-way analysis of variance (ANOVA). Values of In summary, resveratrol had been successfully incorporated into solid lipid nanoparticle in this study. The optimized formulation had a mean particle size of 168.24 ± 10.73 nm with a high zeta potential. Res-loaded SLNs show a remarkable advantage over free resveratrol to induce cancer cell apoptosis and death. Besides, Res-SLNs were observed to have the ability of inhibiting cell invasion. These results indicated that the SLNs could serve as a better carrier for resveratrol to enhance its anti-cancer activity. In-depth biological and in vivo studies will be carried out in our future research.


Because of immune-modulating features, safety, and simple isolation, adipose tissue-derived MSCs (AT-MSCs) are good candidates forsuch studies. However, low availability is the main limitation in using these cells. Different growth factors have beeninvestigated to overcome this issue. In the present study, we aimed to comparatively assess the performance ofAT-MSCs cultured under the presence or absence of three different growth factors, epidermal growth factor (EGF),leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF), following transplantation intesticular torsion-detorsion mice Approximately 1% of the male population suffers from obstructive or non-obstructive azoospermia. Previous This was an experimental study in which AT-MSCs were first isolated from male NavalMedical Research Institute (NMRI) mice. Then, the mice underwent testicular torsion-detorsion surgery and receivedbromodeoxyuridine (BrdU)-labeled AT-MSCs into the lumen of seminiferous tubules. The transplanted cells had beencultured in different conditioned media, containing the three growth factors and without them. The expression of germcell-specific markers was evaluated with real-time polymerase chain reaction (PCR) and western-blot. Moreover,immunohistochemical staining was used to trace the labeled cells. The number of transplanted AT-MSCs resided in the basement membrane of seminiferous tubules significantlyincreased after 8 weeks. The expression levels of Gcnf and Mvh genes in the transplanted testicles by AT-MSCscultured in the growth factors-supplemented medium was greater than those in the control group . The expression levels of the c-Kit and Scp3 genes did not significantly differ from the control group.Our findings showed that the use of EGF, LIF and GDNF to culture AT-MSCs can be very helpful in terms ofMSC survival and localization. All the experiments presented in this study were approved by The Local Animal Care Committees of Ahvaz Jundishapur University of Medical Sciences (AJUMS) (IR.AJUMS.REC.2015.739), which were in complete accordance with the guidelines for the care and use of laboratory animals set by the national academy of sciences . 3 pieces. Blood vessels were removed fromthe tissue as much as possible and the pieces of fat were incubated in collagenase type I enzyme  for 25-30 minutes at 37°C. To stop the enzyme activity, Dulbecco’s Modified Eagle’s Medium  containing 10% fetal bovine serum  was added to the sample. The suspension sample was centrifuged at 1200 rpm for 7 minutes, at room temperature and the cell pellet was cultured in 25 cm2 flasks containing DMEM medium supplemented with 15% FBS and 1% Pen/Strep, and incubated at 37ºC in the presence of 5 % CO2. After three days, the cell medium was replaced with freshmedium and non-cohesive cells were removed. Mediumwas changed once every two days until the cell densityreached 80-90%. The cells were then passaged for more proliferation and purification. For this purpose, 1 ml 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) was added to each flask and incubated for 2-3 minutes. When the cells were floating in the flask, the trypsin was neutralized using 3-4 ml of the medium containing FBS. Then, the cell suspension was centrifuged for 7 minutes. at 1200 rpm and the cell pellet was cultured in new flasks at a density of 20000/cm2  containing 3% penicillin/streptomycin(Pen/Strept) and 0.3% amphotericin B, then cut into1-2 mm0000/cm2  . MSCs are fibroblast analogue cells with adhesion property and differentiation capacity . However-7 M dexamethasone, 50 µg/ml ascorbic acid , and 10 mM B-glycerol phosphate ]. The cells were incubated at 37°C and in 5% CO2 for 21 days. Osteogenic medium was exchanged every 3 days. At the end of the differentiation period, AT-MSCs were fixed with 3% paraformaldehyde and the presence of calcium deposits was examined using 0.5 % alizarin red solution. In order to further characterize of our cultured cells, we assessed their ability to differentiate into osteoblasts and adipocytes. For osteogenic differentiation, AT-MSCs (passage 3) were cultured in a 6-well plate (5×104 cells/well). After 24 hours, the proliferative medium was replaced with osteogenic differentiation medium . The cells were incubated at 37°C and 5% CO2. After 14 days, the cells were fixed with 3% paraformaldehyde and the presence of lipid follicles was examined by Oil Red O staining   , 24. For labeling the AT-MSCs prior to transplantation we used Bromodeoxyuridine (BrdU), which is a base analogue substituted for thymine during DNAsynthesis in proliferating cells. Following the denaturation of double-stranded DNA, BrdU is detected by immunohistochemistry, thus a population of cells that has proliferated is identified . Using tTo create azoospermic mice, we used the testicular torsion-detorsion method. For this purpose, twenty 6-8 week-old male NMRI mice (25-30 g) were first anesthetized by intraperitoneal injection of ketamine and xylazine, then the scrotal midline was cut, tunica vaginalis was opened and the testicle was twisted 720 degrees around its axis in a counterclockwise direction and was fixed with a 4-0 silk suture. Two hours later , the te5 AT-MSCs were injected into the lumen of seminiferous tubules of testicular torsion-detorsion mice by Hamilton syringes. Testicles were fixed in their places and scrotal walls were closed again. The mice were divided into three groups. Group 1 was injected with AT-MSCs cultured in EGF (10 ng/ml), LIF (5 ng/ml), and GDNF (5 ng/ ml) (MSCs-GF group), group 2 was injected with AT-MSCs that were cultured in a medium without growth factors (MSCs-T group), and group 3 was the testicular torsion-detorsion mice that did not receive any cells (negative control). The right testicles of all mice were considered as the positive control group for each treatment. To verify that the injected AT-MSCs have entered the testicles, the cells were stained with trypan blue. 8 weeks after cell transplantation, 5 testicles in each group were removed for molecular analysis. For histological analyses, 3 testicles in each group were removed and fixed in formalin and hematoxylin-eosin staining and immunohistochemical analysis were performed on tissue sections. Six weeks after testicular torsion-detorsion surgery, the mice were anesthetized with ketamine/xylazine, scrotal walls were opened and 10For histological assessment, hematoxylin-eosin stainingwas done. The stages of staining were performed accordingto the standard protocols, as summarized in the study by Cardiff et al. . All reaIn order to trace the AT-MSCs labeled with BrdU, immunohistochemical staining was performed. For this purpose, the tissues containing BrdU were fixed in 4% formalin, then dehydrated and embedded in paraffin. Five-micron thick slices were prepared from the paraffin blocks and placed on slides for immunostaining. The slides were kept at 37°C overnight. Prior to staining, the sections were deparaffinized, then staining was performed according to the BrdU immunohistochemistry kit  instructions. c-Kit, Mvh, Scp3, Gcnf and Gapdh are as follows respectively: The RNeasy Mini Kit  was used to extract the total tissue RNA as per the company instructions. The cDNA was synthesized with Quanti Nova Reverse Transcription Kit  according to the company instructions. Primers for the selected genes were designed specifically using Gene Bank sequences. Primer sequences of c-Kit-F: 5´-GAGAAGGAAGCGTGACTCGT-3´R: 5´-TCTTGCGGATCTCCTCTTGT-3´,Mvh-F: 5´-CGAAACATAGGTGATGAAAGAAC-3´R: 5´-CCACTGAAGTAGCAACAAGAAC-3´,Scp3F: 5´-AAAGCATTCTGGGAAATCTG-3´ R: 5´-GTACTTCACCTCCAACATCTTC-3´,Gcnf-F: 5´-CAACTGAACAAGCGGTATT-3´R: 5´-GATGTATCGGATCTCTGGC-3´, Gapdh-F: 5´-AAGGTCATCCCAGAGCTGAA-3´ R: 5´-CTGCTTCACCACCTTCTTGA-3´.Gapdh gene and data wasevaluated using 2-ΔΔCT approach  stages were performed in Applied Biosystems 7500 Sequence Biosystem. Briefly, 100 nM of the primers and 100 ng cDNA were added to Syber Green PCR master mix to reach the overall volume of 10 µl, then the reaction was carried out in 45 cycles, at 95°C for 15 seconds and 58-60°C for 1 minutes. The gene expression levels in every sample were normalized with the approach . To assess the expression of c-Kit and Gcnf proteins, western blot analysis was performed. In this method, tissue samples were lysed in radioimmunoprecipitation assay buffer (RIPA) solution  supplemented with 0.1% phosphatase inhibitor . The concentrations of the proteins was specified using bicinchoninic acid assay (BCA assay). The equivalent quantity of the protein samples (60 µg) was loaded on 12% polyacrylamide gel, and then transferred to polyvinylidene fluoride (PVDF) membrane . PVDF membrane was blocked at room temperature for one hour in Tris Buffer/Tween 20 (TBST) solution containing 3% skim milk. Then, the membrane was incubated with primary antibodies in the blocking buffer at 4°C overnight: Gcnf , c-Kit , ß-actin . After washing with TBST, the membrane was exposed to the secondary antibody in the blocking buffer [goat anti rabbit IgG-HRP ] for one hour at room temperature. The membrane was then washed in TBST and enhanced chemiluminescence(ECL) western blotting substrate  wasused for detection of the protein bands according to the manufacturer’s instructions. Beta-actin protein was used as a loading control. Image J software was used to measure and compare the density of the proteinbands in the experimental and control groups. For the analysis of the real time PCR tests, the relative expression levels of the genes were calculated by the 2-..CT formula and SPSS version 16  was used for statistical analysis. All quantitative variables were expressed as mean ± SD. The variations were evaluated using one way analysis of variance (ANOVA), Kruskal-Wallis test, Dunnett test and LSD test. For all statistical analyses, the statistical significance was set as P=0.05.AT-MSCs were isolated from the adipose tissue around epididymis of male NMRI mice as was explained before. On the first day, the isolated cells were round shaped, but three days later, the cells became spindle-shaped and fibroblast-like. Other types of cells including endothelial and blood cells were also seen in the flask, however, these cells were eliminated during passaging . Nine days after the induction of adipogenic differentiation,lipid vacuoles within the cells were observed. After 14days, the cells were stained with Oil Red O and the lipidparticles turned red . The firAccording to the results of our previous study, the isolatedcells expressed high levels of CD90 and CD44 markers, andshowed low expressions of CD31 and CD45. These values indicated a high level of purity of the isolated MSCs . Six weeks after torsion-detorsion surgery of mice,hematoxylin-eosin staining of testicle tissue sections wasperformed. In the seminiferous tubules of the testiculartorsion-detorsion mice, most of the sperm cells wereeliminated, spermatogenesis was arrested and the tubuleswere empty from spermatogenic cells, while Sertoli cells andseminiferous tubules structures were maintained . EightwGcnf gene, a germ cell-specificmarker, in both MSCs-GF (P<0.001) and MSCs-T groups(P<0.01) increased significantly compared to the controlgroup. Gcnf gene expression in the MSCs-GF group wassignificantly higher than that in MSCs-T group (P<0.001).The expression level of Mvh another germ cell-specificmarker, in the MSCs-GF group was significantly highercompared to the control group (P<0.05). The expression ofthis gene was not significantly different in the MSCs-T group.The expression levels of Scp3 and c-Kit markers showed no significant difference in either experimental group compared to the control group , but the MSCs-T group showed no significant difference compared to the control group . Stem cell-based therapy has become one of the new potential treatment for the near future in regenerative medicine for the repair of damaged tissues and organs in many diseases such as infertility . AT-MSCsMost of the previous studies show that MSCs confer potential of spermatogenesis recovery in azoospermic animal models , 31-33. in vitro  was significantly higher compared to the control group. Interestingly, MSCs-GF group showed further increased expression of the germ cell-specific markers . It could be due to the impact of the growth factors on viability of the cells. In addition, EGF, LIF, and GDNF are secreted from sertoli cells and testicular niches (c-Kit and Scp3 genes in the AT-MSC recipient groups did not significantly differ from the control group. These genes are related to the final stages of sperm differentiation (Mvh and Gcnf factors in the testicular tissues and reported that c-Kit expression was reduced after 8 weeks. The exact mechanism of function of the transplanted MSCs has not been specified. There are several possibilities in this regard: i. MSCs are differentiated into target tissue cells under the influence of their niche (After 8 weeks, r niches , 21. Thntiation . Howeverntiation  showed ir niche , ii. MSCir niche , and iiiir niche .The observations in the present study may indicatethat the injected AT-MSCs have entered spermatogenicpathway or have revived damaged testicular tissue andSSCs by secretion of trophic factors. To determine its exact mechanism, the cells should be tracked over alonger period of time in the future studies.This study showed that the transplanted AT-MSCs were localized in the basement membrane of seminiferous tubules. The testicles of the mice injected with AT-MSCs expressed spermatogenesis-specific markers. The mice that received cells that were cultured in the presence of growth factors showed overexpression of germ cell-specific markers. According to these results, the use of EGF, LIF and, GDNF to culture AT-MSCs can be very helpful in terms of MSC survival and localization, but further preclinical studies in different animal models and with different time points are needed to develop an effective clinical application.


Introduction: Level of serotonin is mainly regulated by the serotonin reuptake transporter encoded by SLC6A4. The promoter region of SLC6A4 bears a repeat polymorphism 5-HTTLPR and a single nucleotide polymorphism rs25531. We have previously studied the association between these two variants and sporadic PD. The objective of the current study was to determine whether the SLC6A4 polymorphisms were associated with key motor and non-motor symptoms of PD.Methods: A total of 370 PD patients of Han Chinese were included. Associations between the SLC6A4 polymorphisms and PD symptoms including depression, intellectual impairment, tremor and rigidity were analyzed.Results: 5-HTTLPR was associated with depression in PD patients and presence of the LL genotype was protective against the depression risk. The rs25531 was associated with rest tremor in PD and the A allele serves as a recessive risk allele. No associations were found in the two polymorphisms with respect to intellectual impairment and rigidity in the cohort.Conclusion: The current study reveals two PD symptoms associated with SLC6A4 polymorphisms, and provides new insight into how serotonergic system genetically participates in the symptomatic progression of PD. Further study is warranted in additional populations. Parkinson's disease (PD) is a common neurodegenerative disease with bradykinesia, rigidity, and rest tremor as its cardinal motor manifestations . Non-motSLC6A4. There are two polymorphisms in its promoter region, including a repeat polymorphism 5-HTTLPR and a single nucleotide polymorphism rs25531 located within 5-HTTLPR. 5-HTTLPR consists of 14-repeat and 16-repeat variants, as well as several rare variants such as 15-, 19-, 20-, or 22- repeat. The 14-repeat and 16-repeat elements are denoted as the long (L) allele and the short (S) allele, respectively  of Han Chinese ethnicity were included in this study. The median age at onset was 64 . The patients were diagnosed by two movement disorder neurologists according to the UK Parkinson‘s Disease Society Brain Bank Criteria . PatientThe non-motor and motor symptoms were assessed with Unified Parkinson's Disease Rating Scale (UPDRS) by face to face interview and physical examination. Patients were assessed in the practically defined OFF medication state . PatientGenomic DNA was isolated from peripheral blood using a DNA extraction kit . The 5-HTTLPR and rs25531 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism with restriction endonuclease HpaII  as previously described . The priP < 0.05 was considered statistically significant.Data were analyzed using the statistical package of Predictive Analytics Software 18.0  for windows. Kolmogorov-Smirnov test was used for normality test. Differences in age at onset and disease duration between PD subgroups were assessed using Mann-Whitney Test. Differences in gender and genotype frequencies were assessed using Chi square test. Multivariate analysis was performed by binary logistic regression model using stepwise forwards method with gender, age at onset, disease duration, 5-HTTLPR, rs25531, and the interaction between the two polymorphisms as covariates. A two-tailed SLC6A4 in the PD patients. A significant difference (P = 0.023) in 5-HTTLPR was detected between PD patients with and without depression (P = 0.029). Patients carrying the LL genotype had reduced risk toward depression in comparison with the major SS carriers  with the GG genotype being protective compared to the AA (P = 0.032, OR 0.113, 95% CI 0.015–0.831; P = 0.031, OR 0.480, 95% CI 0.246–0.934; Motor symptoms of tremor and rigidity were analyzed to understand their association with SLC6A4 polymorphisms with depression, intellectual impairment, tremor and rigidity, we demonstrate in a currently largest PD cohort that 5-HTTLPR is linked with PD depression and rs25531 is associated with rest tremor.The symptomatology of PD is heterogeneous. Both genetic and environmental factors are believed to be involved in its progression. By analyzing associations of the Extensive evidence have indicated that the 5-HTTLPR is associated with depression risk –19. A coHighlighted should be that we firstly report an association between rs25531 and PD tremor with its A allele as a recessive risk allele. Interestingly, rest tremor is indeed considered to be more likely related to serotonin dysfunction than to dopamine deficiency as suggested by post-mortem and SERT imaging results –30. SeroSLC6A4 polymorphisms are not associated with intellectual impairment and rigidity in the patients. However, our results suggest that the cognitive disturbance is related to disease duration and age at onset. While the role of age at onset appears disputable, it is widely recognized that disease duration is associated with dementia in PD (The ia in PD –39. The ia in PD , and notSLC6A4 polymorphisms in risk toward PD motor and non-motor symptoms. Specifically, our results are in support that 5-HTTLPR modulates PD depression, and for the first time show that rs25531 is associated with rest tremor in PD, thus providing new insight into how serotonergic system genetically participates in the symptomatic progression of PD.In conclusion, the current study suggests a role of J-YW and J-HZ conceived and designed the study. Q-YF, J-YW, XZ, J-HH, and S-GZ collected samples, acquired and analyzed the data. C-PH provided statistical support and analysis. J-YW and J-HZ interpreted the results and drafted the manuscript. XZ and J-HZ supervised the study. All authors have read, revised, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.